Muscle loss is an important medical consequence of spinal cord injury, burns, chronic illness, injury, and aging (Spungen et al., J Appl Physiol 88: 1310-1315, 2000; Yeh et al., Chest 122: 421-428, 2002; Janssen et al., J Am Geriatr Soc 52: 80-85, 2004). The weakness caused by muscle loss reduces mobility and independence, and increases risks of falls and fractures. Muscle loss results primarily from accelerated degradation of muscle proteins by caspases and the ubiquitin-proteasome system (Tiao et al., J. Clin. Invest 94: 2255-2264, 1994; Furuno et al., J Biol Chem 265: 8550-8557, 1990). In this system, proteins are marked by the covalent attachment of the 76 amino acid protein ubiquitin by the formation of an isopeptide bond between the carboxyl terminus of ubiquitin and the ε-amino group of a lysine in the substrate protein (Pickart, Annu Rev Biochem 70: 503-533, 2001). Additional ubiquitin molecules are attached to the previously bound ubiquitin, forming a poly-ubiquitin chain that is recognized by the 26S proteasome. This giant protease complex then degrades the substrate.
Conjugation of ubiquitin to appropriate substrate proteins is catalyzed by E3s (ubiquitin ligases) (Pickart, Annu Rev Biochem 70: 503-533, 2001). The most common form of this ligase is a multimeric complex that includes an E2 (ubiquitin conjugase), one or more proteins providing substrate recognition, and structural proteins. DNA microarray analysis and differential display studies have shown that a gene called Muscle Atrophy F-Box (MAFbx) is greatly upregulated in muscle loss states (Gomes et al., Proc Natl Acad Sci USA 98: 14440-14445, 2001; Bodine et al., Science 294: 1704-1708, 2001; Lecker et al., Faseb J 18: 39-51, 2004). F-Box proteins such as MAFbx are components of SCF (Skp-cullin-F-box) family ubiquitin ligases and may serve important roles in recognizing the substrates for ubiquitination by such complexes. The MAFbx gene is expressed selectively in skeletal muscle and heart suggesting very specific functions in the biology of these tissues (Bodine et al., Science 294: 1704-1708, 2001). Moreover, disruption of the MAFbx gene in mice greatly reduces rates of muscle loss (Bodine et al., Science 294: 1704-1708, 2001). The MAFbx gene is upregulated in all muscle loss states studied to date, including paralysis, starvation, diabetes, renal failure, sepsis, and glucocorticoid excess (Gomes et al., Proc Natl Acad Sci USA 98: 14440-14445, 2001; Bodine et al., Science 294: 1704-1708, 2001; Lecker et al., Faseb J 18: 39-51, 2004; Wray et al., Int J Biochem Cell Biol 35: 698-705, 2003). Consequently, understanding the regulation of MAFbx expression has been of great interest.
Little is known about how expression of this gene is controlled. Muscle specific expression of many other genes is accomplished through muscle differentiation factors such as myogenin and MyoD acting at regulatory elements in promoter regions of such genes (Rawls and Olson, Cell 89: 5-8, 1997). These are transcription factors expressed early in the program of muscle differentiation that continue to be expressed in fully differentiated muscle. Such elements have been found in upstream regulatory regions, in non-coding sequences within the first exon, and in introns (Catala et al., Mol Cell Biol 15: 4585-4596, 1995; Storbeck et al., J Biol Chem 273: 9139-9147, 1998; Wheeler et al., Am J Physiol 276: C1069-C1078, 1999; Smith et al., Am J Physiol 274: C1188-C1195, discussion C1187, 1998; Cheng et al., Endocrinology 143: 4693-4701, 2002; Gilley et al., J Cell Biol 162: 613-622, 2003). Core promoters or their immediate upstream regions also confer tissue selectivity (Smith et al., Am J Physiol 274: C1188-C1195, discussion C1187, 1998). Some insight into how MAFbx expression is upregulated in muscle loss states comes from findings that the forkhead family transcription factor Foxo3A is activated in muscle loss states such as starvation and glucocorticoid toxicity, and that in the mouse MAFbx gene this transcription factor upregulates MAFbx expression by interactions with forkhead transcription factor elements within the upstream promoter and untranslated region of the first exon (Stitt et al., Mol Cell 14: 395-403, 2004; Lee et al., J Am Soc Nephrol 15: 1537-1545, 2004).
The following disclosure elucidates structural and functional attributes of the human MAFbx transcription regulatory sequence, and provides useful compositions and methods based thereon.